Plasmodium berghei EXP-1 interacts with host Apolipoprotein H during Plasmodium liver-stage development.

The first, obligatory replication phase of malaria parasite infections is characterized by rapid expansion and differentiation of single parasites in liver cells, resulting in the formation and release of thousands of invasive merozoites into the bloodstream. Hepatic Plasmodium development occurs inside a specialized membranous compartment termed the parasitophorous vacuole (PV). Here, we show that, during the parasite's hepatic replication, the C-terminal region of the parasitic PV membrane protein exported protein 1 (EXP-1) binds to host Apolipoprotein H (ApoH) and that this molecular interaction plays a pivotal role for successful Plasmodium liver-stage development. Expression of a truncated EXP-1 protein, missing the specific ApoH interaction site, or down-regulation of ApoH expression in either hepatic cells or mouse livers by RNA interference resulted in impaired intrahepatic development. Furthermore, infection of mice with sporozoites expressing a truncated version of EXP-1 resulted in both a significant reduction of liver burden and delayed blood-stage patency, leading to a disease outcome different from that generally induced by infection with wild-type parasites. This study identifies a host-parasite protein interaction during the hepatic stage of infection by Plasmodium parasites. The identification of such vital interactions may hold potential toward the development of novel malaria prevention strategies.

Anti-ß2GPI antibodies enhance atherosclerosis in ApoE-deficient mice.

Accelerated atherosclerosis often occurs in patients with antiphospholipid syndrome (APS), and auto-antibodies to ß2 glycoprotein I (anti-ß2GPI) are confirmed as pathogenic antibodies to APS. Our previous studies have demonstrated that the conversion of mouse peritoneal macrophages into foam cells could be enhanced by co-existence of ß2GPI and anti-ß2GPI IgG, but this phenomenon has not been explored in vivo. Here, we present a mouse model to observe the effect of anti-ß2GPI IgG in the development of atherosclerosis. Male ApoE-deficient mice were intraperitoneally injected with anti-ß2GPI IgG (100 µg/mouse) and homologous control IgG (100 µg/mouse) every week for 16 weeks. Plasma lipid composition, magnetic resonance imaging (MRI) and histological staining were used to evaluate vascular inflammation, lumen stenosis and plaque stability. The results showed that the levels of total cholesterol, triglycerol and low-density lipoprotein-cholesterol in plasma were not changed in all mice fed with high-fat diet, but the level of high-density lipoprotein-cholesterol was lower and the atherosclerosis index was significantly increased in HD + anti-ß2GPI group than in other high-fat diet groups. In addition, compared with NR IgG-treated mice, anti-ß2GPI IgG-treated mice showed more lipid deposition in the carotid artery, markedly narrowed arteriolar lumen as well as higher MMP-9 expression, more macrophages and fewer collagen fibers in the aortic arch root. Furthermore, the aortic mRNA levels of TNF-α, IL-1ß, and MCP-1 were significantly increased in anti-ß2GPI IgG-treated mice. Together, these data indicate that anti-ß2GPI IgG increases vascular inflammation, aggravates atherosclerosis and promotes the formation of vulnerable plaque in ApoE-deficient mice.

Catastrophic antiphospholipid syndrome: Lessons from 14 cases successfully treated in a single center. A narrative report.

The study aimed to evaluate the clinical significance of laboratory findings in patients with catastrophic antiphospholipid syndrome (CAPS) and to report the effects of a well-defined treatment protocol in 14 consecutive cases. Thirteen patients (12 presenting one and one presenting two episodes of CAPS) were consecutively treated and monitored between 1986 and 2017. Antiphospholipid antibody (aPL) characteristics of the patients were compared with those of 64 matched controls (45 antiphospholipid syndrome patients and 19 aPL carriers) who did not develop CAPS during the same mean follow-up period (12 years ±â€¯9.9 SD). Triple aPL positivity (IgG/IgM anticardiolipin + IgG/IgM anti-ß2Glycoprotein I + lupus anticoagulants) significantly prevailed in the CAPS patients with respect to the controls (p = 0.003). IgG anticardiolipin and IgG anti-ß2Glycoprotein I mean antibody titers of the CAPS patients were significantly higher than those of the controls (p = 0.0018 and p = 0.003, respectively). Triple therapy (anticoagulation + plasma exchange + steroids) was administered to all the CAPS cases except for one. Beginning in 2009, intravenous immunoglobulin infusion has also been included in the triple therapy protocol (six patients). All the patients recovered from CAPS; five showed renal failure and one a I-II class New York Heart Association (NYHA) dilated cardiomyopathy. Long-term outcomes of CAPS included a gradual worsening of renal failure in one patient who required hemodialysis 30 years after the acute episode. Renal function improved in the other four patients. The patient affected with dilated cardiomyopathy worsened to a II class NYHA over a five year period. Currently all the patients are alive. A specific antiphospholipid antibody profile could be considered a risk factor associated to CAPS. Early use of a defined treatment protocol based on triple therapy either or not associated with IVIG was associated with recovery in all CAPS patients.

Reversible drug-induced antiphospholipid syndrome.

We report an original case of reversible antiphospholipid syndrome (APS) due to minocycline in a young male patient who experienced recurrent strokes while taking minocycline. He started minocycline therapy (50 mg twice daily) at 15 years old for acne. After three years of treatment, the patient experienced a lateral medullary syndrome. He was treated with aspirin while minocycline was continued. Eighteen months later, the patient complained about horizontal binocular diplopia. MRI revealed an infarct of the oculomotor nerve nucleus. Laboratory investigations revealed high titers of anti-beta 2 glycoprotein 1 (antiß2GP1) antibodies of 470 U/ml (normal range <15 U/ml) and antiphosphatidylethanolamine antibodies of 137.4 U/ml (normal range <18 U/ml). Other laboratory tests were normal. Six weeks after discontinuation of minocycline, anti-ß2GP1 antibodies decreased to 335 U/ml and to 36 U/ml at six months and then remained negative for six years. Many drugs have been considered as possibly causing APS but only in a limited number of patients. To our knowledge this is the first case of drug-induced APS with complete disappearance of high titers of anti-ß2GP1 antibodies after minocycline withdrawal. This case also illustrates the need to monitor the levels of antiphospholipid antibodies, even though initial values are high and confirmed after 12 weeks.

Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-ß2-glycoprotein I (anti-ß2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of ß2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

Validation of a New Panel of Automated Chemiluminescence Assays for Anticardiolipin Antibodies in the Screening for Antiphospholipid Syndrome.

BACKGROUND: Antibodies anticardiolipin (aCL) and anti-ß2-glycoprotein I (aß2GPI) are two of three laboratory criteria of antiphospholipid syndrome (APS). All of assays of antiphospholipid antibodies (aPL), coagulation assays as well as ELISAs, show methodological shortcomings, that affect their sensitivity and specificity. Therefore, we decided to validate these parameters for a new chemiluminescent examination (CLIA). METHODS: aCL and aß2GPI antibodies were measured by ELISAs (AIDA, Bad Kreuznach, Germany) and aß2GPI with CLIA kits (Werfen, Barcelona, Spain). RESULTS: When we evaluated both assays, the coefficient of variation for CLIA was slightly lower (9.04 - 12.74%) than for ELISA (11.05 - 15.3%) and the LOD was 0.2 U/L. The dilution series showed significant linearity for all CLIA methods, aCL IgG, aCL IgM, aß2GPI IgG, and aß2GPI IgM (0 - 3000 U/L), and method comparison studies revealed good agreement with the currently used ELISA (Kappa values ranging 0.534 - 0.936) without determination of aß2GPI IgG. The determination aß2GPI IgG by CLIA method shows higher positivity in 31 samples. These new aCL IgG, aCL IgM, aß2GPI IgG, and aß2GPI IgM tests have excellent analytical characteristics and allow fully automated and simultaneous analysis on an analyzer. CONCLUSIONS: Chemiluminescent determination of an automated analyzer can improve the fundamental parameters of tests such as reproducibility between laboratories.

[Inhibitory effects of fluvastatin on activation of THP-1 cells induced by anti-beta2GPI/beta2GPI complex].

This study is to explore the interventional effects of fluvastatin on anti-beta2GPI/beta2GPI-induced activation in THP-1 mononuclear cells. In vitro, human mononuclear cells THP-1 were treated with fluvastatin, LPS and anti-beta2GPI/beta2GPI, then the TF expression on THP-1 cells was detected by real-time quantitative PCR (RT-qPCR) or TF activity was detected by kit. TNF-alpha mRNA and its protein expression were investigated by RT-PCR and ELISA kit. The expression of phospho-NF-kappaB p65 and inhibitory protein of NF-kappaB (IkappaB-alpha) were measured by Western blotting. The results suggested that the expression of TF and TNF-alpha on THP-1 cells was significantly up-regulated with treatment of anti-beta2GPI/beta2GPI complex (100 mg x L(-1)), compared with that of untreated cells (P < 0.05). Fluvastatin (50 mg x L(-1)) could decrease TF (mRNA and activity) expression and the level of TNF-alpha (mRNA and protein) in THP-1 cells with anti-beta2GPI/beta2GPI complex. The expression of TF and TNF-alpha was shown in a concentration-dependent manner. Moreover, anti-beta2GPI/beta2GPI complex could downregulate IkappaB-alpha levels and increase the levels of phospho-NF-kappaB p65. And these effects of anti-beta2GPI/beta2GPI complex could be blocked by fluvastatin. In conclusion, fluvastatin may interfere the expression and regulation of NF-kappaB signal transduction pathway, thereby inhibit the effects of anti-beta2GPI/beta2GPI on activation of THP-1 cells, by decreasing the expression of TF and TNF-alpha.

Indications for a protective function of beta2-glycoprotein I in thrombotic thrombocytopenic purpura.

It has been shown that ß(2) -glycoprotein I (ß(2) GPI) interacts with von Willebrand factor (VWF) in a glycoprotein (GP)Ib binding state. Given the presence of active VWF multimers in thrombotic thrombocytopenic purpura (TTP), we speculated that ß(2) GPI might play a role in TTP. We found that ß(2) GPI plasma levels were significantly lower in acute and remission TTP patients than in normal controls, showing a direct correlation with ADAMTS 13 levels and an inverse correlation with the extent of VWF activation. In vitro flow experiments demonstrated that ß(2) GPI can block platelet adhesion to endothelial cell-derived VWF strings. We confirmed the direct binding of ß(2) GPI to VWF by surface plasmon resonance, and determined that domain I of ß(2) GPI is the binding site of VWF A1 domain. Adhesion of ß(2) GPI to erythrocytes and platelets was increased in the presence of active VWF, indicating that ß(2) GPI may be cleared from the circulation during TTP episodes together with blood cells. Our findings suggest that ß(2) GPI may protect from the effects of hyper-functional VWF by inhibiting its interaction with platelets.

Immunochemical properties and pathological relevance of anti-ß2-glycoprotein I antibodies of different avidity.

Despite available treatment, there is still significant morbidity and mortality present among patients with the autoimmune thrombophilic condition termed 'antiphospholipid syndrome' (Espinosa, G. and Cervera, R. 2009. Morbidity and mortality in the antiphospholipid syndrome. Curr. Opin. Pulm. Med. 15:413.). High-avidity (HAv) anti-ß(2)-glycoprotein I (anti-ß(2)GPI) antibodies, shown to correlate with thrombotic events in patients, could represent the much needed improved prognostic marker. By studying their effect on crystalline annexin A5 shield on phospholipid surfaces (one of proposed pathogenic mechanisms), with the use of atomic force microscopy, the pathogenic potential of HAv anti-ß(2)GPI antibodies was confirmed. Furthermore, by using surface plasmon resonance and enzyme-linked immunosorbent assays, unique binding characteristics of HAv antibodies in comparison with low avidity antibodies were established. HAv anti-ß(2)GPI were confirmed to (i) recognize ß(2)-glycoprotein I in a solution, (ii) interact predominantly monovalently (much lower dependency on the antigen density) and (iii) form more stable complexes with the antigen. Since enzyme-linked immunosorbent assays currently used in routine diagnostics detect anti-ß(2)GPI antibodies of unknown avidity, our observations are potentially useful for the development of improved diagnostic tests capable of detecting clinically relevant antibodies.

False-positive results in ELISA-based anti FVIII antibody assay may occur with lupus anticoagulant and phospholipid antibodies.

The evaluation of a prolonged aPTT often includes Lupus Anticoagulant, Antiphospholipid Antibodies, and Factor VIII (FVIII) inhibitors. We have noticed that patient samples positive for lupus antibody (LA) are frequently also positive for FVIII IgG antibodies in an enzyme-linked immunosorbent assay (ELISA), indicating the need for follow-up testing with a more labour-intensive functional assay for FVIII inhibition. This study evaluates the potential for a FVIII IgG ELISA to yield false-positive results in patient samples positive for LA or other antiphospholipid antibodies. A total of 289 residual de-identified patient samples positive for LA (n = 143), anti-cardiolipin IgG (n = 84), or beta2-glycoprotein antibody (n = 62) were tested for FVIII IgG using a commercial ELISA. Samples with positive FVIII IgG ELISA results were further tested for FVIII activity using a clot-based FVIII inhibitor assay. The FVIII IgG ELISA yielded positive results in 39 (13%) of the samples tested, including 13/143 (13%) LA-positive, 15/85 (18%) aCL IgG-positive and 6/62 (10%) ß2-glycoprotein IgG-positive samples. The clot-based FVIII inhibitor assay yielded negative results in all 39 FVIII IgG-positive specimens tested, indicating discrepancy with the FVIII IgG ELISA results. Patient specimens positive for LA, aCL IgG, or ß2-glycoprotein IgG may yield false-positive results for FVIII antibodies. Caution is warranted in interpreting FVIII antibody results in these cases.

Toll-like receptor (TLR)-4 mediates anti-ß2GPI/ß2GPI-induced tissue factor expression in THP-1 cells.

Our previous study demonstrated that annexin A2 (ANX2) on cell surface could function as a mediator and stimulate tissue factor (TF) expression of monocytes by anti-ß2-glycoprotein I/ß2-glycoprotein I complex (anti-ß2GPI/ß2GPI). However, ANX2 is not a transmembrane protein and lacks the intracellular signal transduction pathway. Growing evidence suggests that Toll-like receptor 4 (TLR-4) might act as an 'adaptor' for intracellular signal transduction in anti-ß2GPI/ß2GPI-induced TF expressing cells. In the current study, we investigated the roles of TLR-4 and its related molecules, myeloid differentiation protein 2 (MD-2) and myeloid differentiation factor 88 (MyD88), in anti-ß2GPI/ß2GPI-induced TF expressing human monocytic-derived THP-1 (human acute monocytic leukaemia) cells. The relationship of TLR-4 and ANX2 in this process was also explored. Along with TF, expression of TLR-4, MD-2 and MyD88 in THP-1 cells increased significantly when treated by anti-ß2GPI (10 µg/ml)/ß2GPI (100 µg/ml) complex. The addition of paclitaxel, which competes with the MD-2 ligand, could inhibit the effects of anti-ß2GPI/ß2GPI on TLR-4, MD-2, MyD88 and TF expression. Both ANX2 and TLR-4 in THP-1 cell lysates could bind to ß2GPI that had been conjugated to a column (ß2GPI-Affi-Gel). Furthermore, TLR-4, MD-2, MyD88 and TF expression was remarkably diminished in THP-1 cells infected with ANX2-specific RNA interference (RNAi) lentivirus (LV-RNAi-ANX2), in spite of treatment with a similar concentration of anti-ß2GPI/ß2GPI complex. These results indicate that TLR-4 and its signal transduction pathway contribute to anti-ß2GPI/ß2GPI-induced TF expression in THP-1 cells, and the effects of TLR-4 with ANX2 are tightly co-operative.

Effects of anti-ß2-glycoprotein I antibody on PlGF, VEGF and sVEGFR1 production from cultured choriocarcinoma cell line.

AIM: Anti-ß2-glycoprotein I (anti-ß2-GPI) antibody has been detected in cases of recurrent abortion and intrauterine death. Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) are growth factors that accelerate villous proliferation. Soluble VEGF receptor-1 (sVEGFR1) is a soluble receptor for PlGF and suppresses the function of PlGF. In order to study the pathological mechanism of anti-ß2-GPI antibody, we evaluated the effects of anti-ß2-GPI antibody on PlGF, VEGF and sVEGFR1 production from cultured choriocarcinoma cells. METHODS: The sera were taken from six anti-ß2-GPI antibody-positive and six anti-ß2-GPI antibody-negative patients with recurrent miscarriages. Choriocarcinoma cells (JEG-3) were cultured in 24-well plates. After each serum and isolated immunoglobulin G (IgG) were added to the culture medium, the PlGF, VEGF and sVEGFR1 in the culture medium were measured by ELISA 24 h later. When the isolated IgG was added to culture medium, only the levels of PlGF were measured. RESULTS: The anti-ß2-GPI antibody-positive sera and isolated IgG significantly suppressed the production of PlGF from JEG-3 cells more strongly than the anti-ß2-GPI antibody-negative sera. The suppressive effects were not changed by serum inactivation. There was no significant difference in the values of the XTT assay and the production of VEGF and sVEGFR1 between the antibody-positive and antibody-negative sera. CONCLUSIONS: Anti-ß2-GPI antibody-positive sera and IgG suppress the production of PlGF from JEG-3 cells. We suggest that the anti-ß2-GPI antibodies may suppress PlGF production from trophoblasts and cause the failure of placenta formation and function.

In silico identification of new inhibitors for ßeta-2-glycoprotein I as a major antigen in antiphospholipid antibody syndrome.

Beta 2 glycoprotein I (ß2GPI) is a major antigen for autoantibodies present in antiphospholipid antibody syndrome (APS). ß2GPI is a single polypeptide with five repeated domains and different conformations. The activated J-shaped conformation of ß2GPI binds to negatively charged phospholipids in the membrane via the fifth domain and causes blood clotting reactions. We applied a drug repurposing strategy using virtual screening and molecular dynamics to find the best FDA drugs against the fifth domain of ß2GPI. In the first phase, FDA drugs that had the most favorable ΔG with the fifth domain of ß2GPI were selected by virtual screening. Among these drugs that had the most favorable ΔG, Vorapaxar and Antrafenine were selected for molecular dynamics (MD) simulation studies. MD simulation was performed to evaluate the stability of Vorapaxar and Antrafenine complexes and the effect of the two drugs on protein conformation. Also, MD simulation was done to investigate the effect of Antrafenine and Vorapaxar on the binding of ß2GPI to the platelet model membrane. According to the results, Vorapaxar and Antrafenine were bound to the protein with the favorable binding energy (Vorapaxar and Antrafenine binding energies are - 49.641 and - 38.803 kcal/mol, respectively). In this study, it was shown that unlike protein alone and protein in the Antrafenine complex, the protein in the Vorapaxar complex was completely separated from the model membrane after 350 ns. Moreover, Vorapaxar led to more changes in the activated J-shape of ß2GPI. Thus, Vorapaxar can be a suitable candidate for further investigations on the treatment of APS.

Insights into the pathogenesis of catastrophic antiphospholipid syndrome. A case report of relapsing catastrophic antiphospholipid syndrome and review of the literature on ischemic colitis.

We present the case of a woman with a severe clinical history of antiphospholipid syndrome and persistent positivity for lupus anticoagulant, IgG anticardiolipin and IgG anti-ß2Glycoprotein I antibodies. An acute clinical onset characterized by severe abdominal pain immediately followed by circulatory shock and histological colonic small vessel thrombosis pattern pointed to a diagnosis of ischemic colitis. The subsequent rapid onset of pulmonary alveolitis and heart failure associated to subendocardial hypoperfusion led to a diagnosis of definite catastrophic antiphospholipid syndrome (CAPS). Conventional triple therapy together with a broad-spectrum preventive antibiotic therapy were quickly initiated, and the outcome was favorable. We evaluated the patients with ischemic colitis in CAPS described in the literature between 1992 and May 2019 and our CAPS case. In accordance with the "two-hit" hypothesis and on the basis of the patients' data, we would like to speculate that the colonic wall necrosis related to ischemic colitis damaged the intestinal barrier causing loss of resistance to bacteria and leading to endotoxemia and bacteremia with bacteria translocation through the circulatory stream to the lungs and heart. The bacteria acted as the priming factor which favored the binding of ß2Glycoprotein I to the endothelium vessels in the colon, lungs, and heart following activation of anti-ß2Glycoprotein I antibodies which attached to the domain I of ß2Glycoprotein I. This was followed by complement activation which triggered the thrombotic and cytokine storm. If further clinical studies confirm this hypothesis, the treatment of CAPS could be more targeted and effective.

Distinct and overlapping effects of ß2-glycoprotein I conformational variants in ligand interactions and functional assays.

One of the most abundant coagulation proteins is ß2-glycoprotein I (ß2GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) ß2GPI and converted it into an open conformation. The effectiveness of these procedures was assessed by Western blot and negative-staining electron microscopy. We found that in coagulation assays the open form of ß2GPI had a significant prolonging effect on fibrin formation in a dilute prothrombin time test (p < 0.001). In the dilute activated partial thromboplastin time test, both conformations had a significant prolonging effect (p < 0.001) but the open conformation was more effective. In a fluorescent thrombin generation assay both conformations slightly delayed thrombin generation with no significant effect on the quantity of formed thrombin. By using surface plasmon resonance assays, the equilibrium dissociation constants of both the open and closed conformations of ß2GPI showed a similar and strong affinity to isolated anti-ß2GPI autoantibodies (Kd closed ß2GPI = 5.17 × 10-8 M, Kd open ß2GPI = 5.56 × 10-8 M) and the open form had one order of magnitude stronger affinity to heparin (Kd = 0.30 × 10-6 M) compared to the closed conformation (Kd = 3.50 × 10-6 M). The two different forms of ß2GPI have distinct effects in functional tests and in ligand binding, which may considerably affect the intravascular events related to this abundant plasma protein in health and disease.

Laboratory classification categories and pregnancy outcome in patients with primary antiphospholipid syndrome prescribed antithrombotic therapy.

BACKGROUND: A relationship between antibody profile and pregnancy outcome in patients with a previous diagnosis of primary antiphospholipid syndrome (APS) has not been clearly documented. METHODS: Women attending our Center with primary APS characterized by the presence in the blood of one or more of the following: Lupus Anticoagulant (LA), IgG/IgM anticardiolipin (aCL), IgG/IgM anti-human beta2-Glycoprotein I (abeta2GPI) antibodies (confirmed after a minimum of 3 months) were considered eligible for this study. Women who became pregnant during the study period with the exception of those with congenital thrombophilia or other congenital abnormalities were included in our analysis. Primary outcome events, defined as early abortion or fetal death, were evaluated in relation to the laboratory classification category assigned to each patient at the time they were diagnosed with APS. RESULTS: A total of 97 pregnancies occurring in 79 primary APS patients during the study period were analyzed. Twelve out of 97 pregnancies were unsuccessful, 11 out of 65 (16.9%) in category I patients (more than one positive laboratory test) and 1 out of 32 (3.1%) in category II patients (single positive test; adjusted hazard ratio 1.9; 95% CI, 0.2 to 18.9, p=0.6). Pregnancy loss took place in 10 out of 19 pregnancies (52.6%) in women belonging to category I with triple positivity and in 1 out of 46 pregnancies (2.2%) in patients with double positivity. The rate of pregnancy loss was more frequent in the 19 pregnancies of patients with triple positivity than in the 46 pregnancies of double positive patients (adjusted hazard ratio 23, 95% CI, 1.3 to 408, p=0.03). CONCLUSION: Poor pregnancy outcomes occur more frequently in category I than in category II primary APS patients. However, it has been seen that a greater predictability is achieved when category I patients are grouped into triple and double positivity states.

Effects of ß2/aß2 on oxLDL-induced CD36 activation in THP-1 macrophages.

AIMS: ß2-glycoprotein I/anti-ß2-glycoprotein I antibody complex (ß2/aß2) could promote oxLDL-induced endothelial inflammation through Toll-like receptor 4 (TLR4), therefore accelerates atherosclerosis in patients with anti-phospholipid syndrome (APS). However, effects of ß2/aß2 and TLR4 on oxLDL-induced CD36 activation in macrophages remain to be elucidated and are currently under investigation. MATERIALS AND METHODS: THP-1 macrophages with or without the pre-treatment of TAK-242, a TLR4 inhibitor, were treated with RPMI 1640, oxLDL, oxLDL+ß2/aß2 or oxLDL + LPS.CD36 expression and subsequent intracellular lipid accumulation, cholesterol-transportation-related proteins (ACAT1, ABCG1 and ABCA1) expression, inflammatory cytokines (IL-1ß, TNF-α and IL-6) secretion, focal adhesion kinases (FAK) activation and matrix metalloproteinases (MMP-2 and MMP-9) expression by these THP-1 macrophages were evaluated. Moreover, effects of TLR4 on oxLDL+ß2/aß2-induced peroxisome proliferators-activated receptor-γ (PPAR-γ) expression and CD36 translocation have also been observed. KEY FINDINGS: Compared with oxLDL-treated ones, CD36 expression, intracellular lipid accumulation and FAK activation were inhibited, whereas the levels of inflammatory cytokines and MMPs were upregulated in THP-1 macrophages treated with oxLDL+ß2/aß2 (p < 0.05). Moreover, observed differences between oxLDL-treated and oxLDL+ß2/aß2-treated THP-1 macrophages could be reversed by TAK-242 pre-treatment (p < 0.05). Furthermore, oxLDL+ß2/aß2 promoted PPAR-γ expression and CD36 cytoplasmic translocation in THP-1 macrophages, these effects could also be attenuated by TAK-242 (p < 0.05). SIGNIFICANCE: Through a TLR4 dependent manner, ß2/aß2 inhibited oxLDL-induced CD36 expression, lipid accumulation and FAK activation, while promoted inflammatory cytokines and MMPs expression in THP-1 macrophages, indicating the novel dual roles played by ß2/aß2 in APS-related atherosclerosis.

Relationship and significance between anti-beta2-glycoprotein I antibodies and platelet activation state in patients with ulcerative colitis.

AIM: To study the relationship between anti-beta2-glycoprotein I (abeta2GPI) antibodies and platelet activation state in patients with ulcerative colitis (UC) and its significance. METHODS: Peripheral blood samples were collected from 56 UC patients (34 males and 22 females, aged 43.5 years, range 21-66 years), including 36 at active stage and 20 at remission stage, and 25 sex-and age-matched controls. The level of abeta2GPI was measured by ELISA. The platelet activation markers, platelet activation complex-I (PAC-I) and P-selectin (CD62P) were detected by flow cytometry. RESULTS: The A value for IgG abeta2GPI in the active UC group was 0.61 +/- 0.13, significantly higher than that in the remittent UC and control groups (0.50 +/- 0.13 and 0.22 +/- 0.14, P < 0.01). There was a significant difference between the two groups (P < 0.01). The A value for IgM abeta2GPI in the active and remittent UC groups was 0.43 +/- 0.13 and 0.38 +/- 0.12, significantly higher than that in the control group (0.20 +/- 0.12, P < 0.01). However, there was no significant difference between the two groups (P > 0.05). The PAC-I positive rate for the active and remittent UC groups was 30.6% +/- 7.6% and 19.6% +/- 7.8% respectively, significantly higher than that for the control group (6.3% +/- 1.7%, P < 0.01). There was a significant difference between the two groups (P < 0.01). The CD62P positive rate for the active and remittent UC groups was 45.0% +/- 8.8% and 31.9% +/- 7.8% respectively, significantly higher than that for the control group (9.2% +/- 2.7%, P < 0.01). There was a significant difference between the two groups (P < 0.01). In the active UC group, the more severe the state of illness was, the higher the A value for IgG abeta2GPI was, and the positive rate for PAC-I and CD62P was positively correlated with the state of illness (Fabeta2GPI = 3.679, P < 0.05; FPAC-I (%) = 5.346, P < 0.01; and FCD62P (%) = 5. 418, P < 0.01). Meanwhile, in the same state of illness, the A value for IgG abeta2GPI was positively correlated to the positive rates for PAC-I and CD62P. CONCLUSION: abeta2GPI level, platelet activation state and their relationship of them are closely correlated with the pathogenesis and development of UC.

Further Investigations of the Effects of Anti-ß2GP1 Antibodies on Collagen-Induced Platelet Aggregation.

Anti-beta-2-glycoprotein 1 (anti-ß2GP1) antibodies are associated with increased thrombotic risk in patients with autoimmune disease. There is conflicting evidence on the effects of anti-ß2GP1 antibodies on platelets, with both enhanced and inhibited aggregation previously reported. However, previous studies did not include isotype antibodies to ensure the observed effects were due to anti-ß2GP1 antibodies. The aims of this study were to (1) investigate the effects of anti-ß2GP1 antibodies on collagen-induced platelet aggregation in parallel with negative control (buffer normal saline) and isotype control antibodies and (2) determine the lupus anticoagulant (LA) activity of anti-ß2GP1 antibodies used. Three animal-derived anti-human-ß2GP1 antibodies (1.25, 2.5, and 5 µg/mL) incubated with healthy platelet-rich plasma were activated by collagen (2.5 µg/mL). Each anti-ß2GP1 antibody demonstrated the inhibition of aggregation compared to negative control, but not to isotype control. No anti-ß2GP1 antibody demonstrated LA activity, suggesting they were probably nonpathological. This study highlights both negative and isotype control markers are important to validate the effects of anti-ß2GP1 antibodies. Assays to measure anti-domain I-ß2GP1 antibodies are recommended to be used in conjunction with functional measures to further investigate the effects of anti-ß2GP1 antibodies.

Anti-ß2GPI/ß2GPI induces neutrophil extracellular traps formation to promote thrombogenesis via the TLR4/MyD88/MAPKs axis activation.

Antiphospholipid antibodies (aPLs) are a large group of heterogeneous antibodies that bind to anionic phospholipids alone or in combination with phospholipid binding proteins. Increasing evidence has converged to indicate that aPLs especially anti-ß2 glycoprotein I antibody (anti-ß2GPI) correlate with stroke severity and outcome. Though studies have shown that aPLs promote thrombus formation in a neutrophil-dependent way, the underlying mechanisms remain largely unknown. In the present study, we investigated the effect of anti-ß2GPI in complex with ß2GPI (anti-ß2GPI/ß2GPI) on neutrophil extracellular traps (NETs) formation and thrombus generation in vitro and in vivo. We found that anti-ß2GPI/ß2GPI immune complex induced NETs formation in a time- and concentration-dependent manner. This effect was mediated by its interaction with TLR4 and the production of ROS. We demonstrated that MyD88-IRAKs-MAPKs, an intracellular signaling pathway, was involved in anti-ß2GPI/ß2GPI-induced NETs formation. We also presented evidence that tissue factor was expressed on anti-ß2GPI/ß2GPI-induced NETs, and NETs could promote platelet aggregation in vitro. In addition, we identified that anti-ß2GPI/ß2GPI-induced NETs enhanced thrombus formation in vivo, and this effect was counteracted by using DNase I. Our data suggest that anti-ß2GPI/ß2GPI induces NETs formation to promote thrombogenesis via the TLR4/MyD88/MAPKs axis activation, and could be a potentially novel target for aPLs related ischemic stroke.